The Determination of Total Dissolved Nitrogen in Streamwater
Modification of 4500-Norg D (Standard Methods for the Examination of Water and Wastewater- APHA 1995, 19th edition). This is a modification of the method used by Neil Bettez (also some modifications by S. Thomas and Adrian Green). The persulfate concentration and type of acid were changed.
This method measures the amount of total dissolved nitrogen (NH4 +, NO3-, and organic N) in a water sample. The original method assumes that the sample is NOT acidified, however, modifications were made to the amount of NaOH added to account for sample acidification (ACG 1/03). A 10mL sample is digested by autoclaving the sample and a persulfate solution. The digestion causes all forms of N to be oxidized to NO3-. The pH of the sample is then readjusted with acid and analyzed on the Alpkem or Lachat for NO3- (which is the concentration of TDN in the sample). Care must be taken to ensure complete digestion. Organic reference materials must be used to check that the persulfate digestion has gone to completion. The standards and reference materials get digested in the exact same manner as the samples.
· Measure 10mL of standard/sample into a clean, pre-calibrated, black-capped (teflon liner) digestion tube
· Add 2mL K2S2O8 solution (0.05g/mL—see note below!)
· Add 0.23 mL of 5.6M NaOH*
· Cap TIGHTLY and shake
· Autoclave for 1 hour
· Let cool and add 0.08 mL (80 ml) of 5.6N HCL** to each sample (if sample will be analyzed right away)
· Transfer samples to glass scint vials and determine N concentration on autoanalyzer.
· Leave samples in digestion tubes and analyze on the Lachat directly.
*original method calls for 0.21 ml NaOH. Since our samples were acidified in the field with 0.1ml of 6N HCL per 50 ml of sample, we adjusted the volume of NaOH in order to bring the pH to 6-7.
**original method calls for 0.4 ml of 0.9N H2SO4. We chose to use HCL, since that is what our samples were acidified with initially.
Add 224 g NaOH slowly to DI in a volumetric placed in an ice bath. When all NaOH is dissolved and volumetric has returned to room temperature, qs to 1L. Store NaOH in a plastic container.
Make only as much K2S2O8 as you will need for one day, as it degrades over time. Add 5g K2S2O8 per 100mL DI and stir until dissolved (with a little bit of heat; it will take some time to dissolve)
NOTE: The K2S2O8 used is low P and low N persulfate from JT Baker (VWR# JT3239-1).
Add 25 mL concentrated H2SO4 to DI and qs to 1L
NOTE: Make fresh to avoid NH4 + absorption (i.e., don't use that old stuff you made months ago).
Add 460 mL of 12.1 N HCL to DI and bring up to IL with DI.
1,000µM N/L Stock
Weigh 0.101g dried KNO3 and add to a 1L volumetric
dissolve completely in DI
bring up to 1L with DI
transfer to an acid-washed bottle, add a few drops of chloroform, and store in the refrigerator
Bring the stock 1,000µM N/L stock solution to room temperature
100µM N/L working standard - Dilute 10 ml of 1000 µM N/L stock in 100 ml DI
0µM- DI only
1µM- 1mL of 100µM and dilute with DI to 100mL (1*100/100 = 1)
or, 0.1 ml of 1000µM brought to 100 mL with DI
2µM- 2mL of 100µM and dilute with DI to 100mL (2*100/100 = 2)
or, 0.2 mL ml of 1000µM brought to 100 mL with DI
5µM- 5mL of 100µM and dilute with DI to 100mL (5*100/100 = 5)
or, 0.5 mL ml of 1000µM brought to 100 mL with DI
notes: Since our samples were acidified in the field, we acidified our standards to the same Normality as the samples.
We digested a standard curve along with the samples, and used the digested curve to calculate sample concentrations. (ACG)
Standard Reference Materials
Use one or more of the below organics to check that the persulfate is converting organic N to inorganic N. It is best to make one reference standard at the highest level of standard you're using and then another that's about the average concentration of your samples (to check digestion efficiency at the far range and to check the standard curve in the range where most of the samples are).
Nicotinic Acid p-toluenesulfonate (C13H13O5SN)
This is supposed to be a difficult organic standard to digest and is ideal for a digestion check standard.
Make a primary standard by dissolving 0.2953g nicotinic acid p-toluesulfonate in DI. qs to 1L.
Dilute to the proper standard and sample range.
Make a primary standard of 4,000µM (4mM) by dissolving 0.278g nitrophenol in 500mL DI.
Make a working 40µM standard by diluting 1mL of 4,000µM in 100 mL DI.
(This is used as a SRM for TDP but may also be used for TDN as well.)
Make a primary 2,000µMP standard by dissolving 0.1835g ATP in 500mL
Make a working 33.3µMP standard by dissolving 1mL 2,000µM standard in 100mL
NOTE: This working standard also contains 33.3µMN
(NOTE: but ATP is 7% water, so solution is not 33.3µM, but actually 31µM)
Fisher makes a QC standard that contains N and P forms that can be used to check the accuracy of the standards. This reference material will NOT challenge the digestion and should not be used to check the digestion efficiency, but it is a helpful reference material to check the accuracy of your standards.
Screw top test tubes Fisher #14-930-10E
Additional teflon-lined caps Fisher # 14-930-15E
Potassium Persulfate JT Baker (VWR# JT3239-1)
(note: this cannot be substituted with a cheaper persulfate!!)
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